1. Field of the Invention
The present invention is directed to new and useful processes for the large scale production of thio.analogues of phosphonoformic acid (PFA) and to the conversion of phosphonates into thiophosphonates in general as well as to the thio-PFA (TPFA) compounds produced by these procedures. An additional aspect of the present invention relates to the use of these thio-PFA compounds as antiviral agents which are particularly effective against HIV.
2. Description of the Prior Art
Organic compounds of the general structure ##STR1## wherein X is oxygen (O) or sulphur (S) are known, respectively, as phosphonates and thiophosphonates. These compounds are implicated in a variety of biological processes and show promise in basic research for medical and agricultural uses including pesticides and antiviral compounds. Unfortunately, research involvingthiophosphonates is often hindered by the extreme difficulty in producing even small quantities of these phosphonate analogues. Moreover, economic methods for the large scale production of thiophosphonates are virtually unknown.
For example, of particular interest to the present invention are the thio-analogues of phosphonoformic acid. Phosphonoformic acid (PFA) and its thio-analogue. thiophosphonoformic acid (TPFA) have the following general formulae and structures: ##STR2## Early efforts reportedly producing TPFA utilized the Michaelis-Becker reaction between the sodio-derivative of diethyl thiophosphite and ethyl chloroformate or chloroacetate, followed by the removal of the P-OEt groups with iodotrimethylsilane (ITMS) at high temperature over 48 hours. However, recent research has indicated that this method is not reproducible and, because of difficulties including the removal of the ethyl groups, produces mixtures of a variety of compounds rather than the desired TPFA. Other proposed methods for the synthesis of TPFA are equally difficult and expensive utilizing numerous steps with exceptionally lowproduct yields. Similar difficulties and expense are associated with the production of the thio-analogues of other phosphonates as well.
Difficulties in producing usable quantities of thiophosphonates are not restricted to commercial applications requiring large quantities of product. Basic research involving these compounds also requires readily available, pure materials. For example, as proposed and claimed by the present invention, TPFA shows great promise as an antiviral agent for use in combatting HIV infection and AIDS in mammals. These properties could not be determined in the past due to the inability of the prior art methods to produce usable quantities of essentially pure TPFA. However, before discussing these antiviral properties in detail, a general understanding of antiviral therapy will be of assistance.
Unlike infectiousbacteria, which are functionally and physically distinct and can reproduce outside the cells of their host organisms, the simplicity of viruses makes them able to replicate only by physically invading a host cell and co.opting its biochemical mechanisms to make new viral components. As a result of this intimate connection with the replication cycle of the host cell, viruses present few unique biochemical features which can be selectively attacked without poisoning the host cell. As recently as the 1960's, it was believed that the only strategy for controlling viral infections was the development of vaccines against specific viruses to forestall infection by stimulating the immune system of uninfected individuals in advance.
In spite of these problems, recent developments in the understanding of the details of viral functions have brought to light unique aspects of viral activities which may provide targets for attack. This accumulating body of knowledge has made it possible to identify compounds that may selectively interfere with these viral activities without poisoning the host cells of the infected organism. For example, in both lytic viral infections (those that spread rapidly throughout the population of vulnerable cells, destroying them early in the illness) and persistent viral infections (those that do not always kill an infected cell) the viral agents complete their replication cycles through a number of unique steps that an antiviral drug may interrupt.
Unfortunately, the present state of the art is such that antiviral drugs are only capable of attacking such viruses when they are replicating. Attacking a latent virus such as HIV which does not reproduce itself following infection until reactivated by presently unknown factors wouldrequire distinguishing the viral genetic material from the surrounding host genetic material and selectively destroying it. Thus. the current generation of antiviral drugs is only effective against replicating viruses.
Nonetheless, there are notable successes in the field of antiviral drug therapy. An exemplary antiviral compound is acyclovir, a nucleoside analogue which mimics the structure of a precursor of DNA Acyclovir has been found to interfere with the viral enzymes thymidine kinase and DNA polymerase specific to some herpes viruses, thereby inhibiting the synthesis of the viral DNA and ultimately viral replication itself. Similar antiviral effectiveness has been produced with a different nucleoside analogue. ribavirin which interferes with a viral enzyme crucial to the synthesis of DNA and RNA as well as selectively inhibiting viral mRNA and thus the production of viral proteins. Though far more effective against viral functions, ribavirin, like many antiviral compounds, may also affect human cells and thus may be toxic to rapidly metabolizing cells such as blood cells, limiting its applicability and usefulness.
In spite of these and other antiviral success stories, the most important current challenge for the development of antiviral compounds is the need for an effective treatment against HIV, the viral cause of the AIDS pandemic. In contrast to the bleak epidemiological picture of AIDS wherein potentially millions of people are believed to be infected, the accumulation of knowledge aboutHIV and its functions has been unprecedentedly rapid. Though only identified in 1983, HIV is known to be a retrovirus whose main target is the T4 lymphocyte, a white blood cell which marshals the immune defenses of the infected host. Additionally, the virus also infects cells in the central nervous system.
After binding to a host cell, HIV penetrates the cell and exposes its viral genetic material: a single strand of RNA. Accompanying the viral RNA is a viral enzyme known as reverse transcriptase which converts the viral genetic material into DNA which becomes integrated into the chromosomes of the infected host cell. The integrated viral genome or "provirus" remains latent until the host cell is stimulated and then directs the synthesis of viral proteins andRNA which assemble to form new HIV particles which burst from and destroy the host cell.
The current target for antiviral drug therapy against HIV replication is the reverse transcription step which is crucial to the viral replication yet irrelevant to the infected host cells. A variety of antiviral drugs have been shown to reduce the activity of HIV reverse transcription in vitro to varying degrees. These compounds include azidothymidine (AZT), suramin, antimoniotungstate, dideoxynucleotides, and phosphonoformate AZT, has shown significant positive effects in large.scale clinical trials though major concerns remain about its considerable toxicity to bone.marrow cells.
Several researchers have indicated that the pyrophosphate analogues. phosphonoacetic acid (PAA) and phosphonoformic acid (PFA) possess antiviral properties in that they inhibit the replication of several viruses including influenza virus A and herpes virus HSV.I. Research has shown that these compounds have an inhibitory activity on the reverse transcriptase of influenza virus A and the DNA polymerase of HSV.I as well as on the DNA polymerase of mammalian cells. (D. W. Hutchinson, G. Semple. and D. M. Thornton. Synthesis and Biochemical Properties of Some Pyrophosphate Analogues, Biophosphates and Their Analogues Synthesis, Structure, Metabolism and Activity, K. S. Bruzik and W. J. Stec (Eds.). Elsevier Science Publishers, B. V., 1987, 441.450.)
Additionally, it has also been suggested in the art that the thio-analogues of phosphonoacetic acid (PAA) and phosphonoformic acid (PFA) may have potential as antiviral agents. (D. W. Hutchinson and S. Masson. The antiviral potential of compounds containing the thiophosphoryl group. I.R.C S. Medical Science 14 (1986) 176.177.) However, recent research by the inventor has raised significant questions as to the veracity of such reports. It is believed that the reported activities of the alleged thio-PFA compounds discussed in these prior art references are deceivingly incorrect as the prior art methods for preparing these compounds do not produce TPFA but, instead, produce mixtures of different, unidentified compounds.
Moreover, as those skilled in the art will appreciate, further questions as to the accuracy and basis of such unsupported speculation with respect to the proposed properties of TPFA results from the fact that the inhibition of viral enzymes by such compounds in general is uniquely specific to the viral enzymes involved. Thus, it is impossible to predict the antiviral activity of a particular compound as that compound may or may not be effective against a particular virus. For example. acyclovir has proven to be beneficial in infection by Herpes virus, yet acyclovir.resistent strains of Herpes virus have been located. Similarly, Epstein-Barr virus (EBV) is relatively insensitive to acyclovir. Thus, it is clear that early signs of some antiviral activity are not indicative of a compound's effectiveness as an antiviral drug.
Further complicating matters, a compound which may inhibit viral activity may also inhibit critical functions of the host cell and thus prove to be toxic to the host. As a result, antiviral compounds which may be effective in vitro may not be effective as antiviral agents in vivo due to a lack of significant differences in their relative inhibitory activities with respect to viral and host cell mechanisms.
Accordingly. it is a principal object of the present invention to disclose methods for the effective production of large quantities of thio-analogues of PFA in order to facilitate the research and utilization of such compounds.
It is an additional object of the present invention to disclose Processes for inexpensively producing large quantities of relatively pure TPFA and its analogues.
It is a further object of the present invention to disclose novel thio-analogues of PFA.
As those skilled in the art will also appreciate, it is also an object of the present invention to disclose novel methods for converting the general class of phosphonate compounds into thiophosphonates in a simple and economical manner.
It is yet another object of the present invention to disclose methods for inhibiting viral and viral enzyme activities, including those of HIV, utilizing TPFA.
Lastly, it is a further additional object of the present invention to disclose methods for treating HIV infection in mammalian cells utilizing TPFA or its addition salts as effective antiviral compounds.